This invention relates to processes for producing small particles of biological molecules at low temperatures using ultrasonic energy.
It is frequently desirable to incorporate biologically active proteins or other molecules within a polymeric matrix for use in controlled drug delivery or other applications requiring stable, uniform dispersions of material. Reduction of the particle size of such substances, particularly those that have biological activity such as proteins, is an important part in the design of pharmaceutical formulations.
For example, in the preparation of injectable polymeric microspheres for controlled drug delivery systems, the microspheres should be 50 micrometers or less in diameter. In order to achieve a homogeneous distribution of drug particles throughout the matrix of the microsphere, the particles should have diameters much smaller than that of the microsphere itself. Smaller diameter particles loaded in such a system will result in more desirable release kinetics and can help eliminate the initial large amount of drug that is often released (the xe2x80x9cburst effectxe2x80x9d) from these systems. By controlling the particle size of biological molecules in a controlled release device, one can also vary the release rate of drug from these systems, as described by Brown, et al., in xe2x80x9cControlled Release of Insulin from Polymer Matricesxe2x80x9d, Diabetes, 35, 684-691 (1986).
Most small particles of compounds to be used for pharmaceutical applications are prepared using conventional techniques such as spray dying, milling, grinding and lyophilization and sieving. Each of these techniques can reduce the particle size of the materials of interest, but the diameters are often not small enough for controlled release microsphere systems. These methods also have other disadvantages. Spray drying can reduce particles to sizes of five micrometers or less, but biologically active proteins can become inactivated in the process due to denaturation at the aqueous air interface and by exposure to heat generated to evaporate the solvent. Spray drying is also inefficient and much material is lost due to sticking to the large surface area of the apparatus.
Milling and grinding can produce particles having diameters of only five micrometers, however, many of the particles are much larger in size. Because milling and grinding both require large amounts of material, these techniques are not very amenable to expensive biologically active proteins which can often be obtained only in small amounts. Milling and grinding can also result in denaturation of proteins. Sieving of lyophilized powders results in particle sizes of about 70 micrometers or greater.
It is therefore an object of the present invention to provide a method for producing small particles of biological molecules.
It is a further object of the present invention to provide particles of biologically active molecules that can be uniformly dispersed in a polymeric matrix for controlled release of the biologically active substance.
A method for producing small particles of biologically active molecules ranging in diameter from approximately 0.1 to ten micrometers which retain greater than 70% to 95% of their original biological activity, wherein the yield of particles of the biologically active molecules is 80% or greater. The technique can be used to reduce the particle size in preparations of biologically active molecules, including macromolecules such as proteins or smaller molecules such as steroids, amino acids, or synthetic drugs, while maintaining the activity of the molecules. The process can also be used to reduce the particle size of other molecules that are not biologically active, such as sugars or mixtures of these molecules with proteins.
The process for reducing the particle size of these compounds involves two principal steps. In the first step the molecules of interest are dissolved in a solvent and atomized into a low temperature liquified gas. The liquified gas is then evaporated, leaving frozen spherical particles containing solvent and the material of interest. The solvent is removed from the frozen spheres by lyophilization, resulting in the formation of porous spheres having diameters in the range of 10 to 60 micrometers. These spheres can be used as is, if larger particles are desired, or, if smaller particles are desired, they can be suspended in a non-solvent and exposed to ultrasonic energy or other mechanical forces to break the spheres into smaller particles having diameters in the range of from about 0.1 to 10 micrometers. The porous spherical particles or the smaller particles can be suspended in polymer or pharmaceutical carrier-solvent solutions and entrapped for use in drug delivery systems since the particles can be homogeneously dispersed throughout the matrix, resulting in reproducible and controllable release kinetics.